Four Holstein-Friesian dairy cows, fitted with ruminal and duodenal cannulas, were used to evaluate the effect of 0 and 300 mg/d monensin offered with a total mixed ration (TMR) on rumen fermentation, site of nutrient digestion, and milk yield and composition. Indigestible markers chromium EDTA and ytterbium acetate were ruminally infused and their concentration measured at the duodenum to determine digesta flow rates. Choice of method of processing digesta samples caused large differences in the variation in digesta flow calculations. Monensin increased rumen molar proportions (mmol/total mol) of propionic acid (214 vs 268) and reduced those of acetic (622 vs 583) and n-butyric acid (134 vs 119). The values recorded for monensin treated cows for nitrogen (N) digested in the rumen, flow of total N and non-ammonia N (NAN) to the duodenum and N voided in the faeces were 5.6, 6.1, 7.1 and 16.0% units lower compared with the control. Monensin decreased starch digestion in the rumen (9.4% units), increased starch flow to the duodenum (0.5 kg/d) but did not alter total tract digestion. Monensin had no significant effect on rumen or total tract digestion of organic matter (OM), acid detergent fibre (ADF) and nitrogen. Milk yield was increased numerically by 1.9 kg/d, and milk fat and milk protein contents were reduced by 3.9 and 1.5 g/kg, respectively, but yields of fat and protein were unaffected.